PRP (platelet-rich plasma) combined carboxymethyl chitosan scaffold as well as preparation method and application thereof

Abstract

The invention discloses a PRP combined carboxymethyl chitosan scaffold and a preparation method and application thereof, and belongs to the technical field of biomaterials.The preparation method comprises the following steps that carboxymethyl chitosan powder and hydroxyapatite powder are mixed, genipin powder is added, the mixture is mixed to be uniform, and a mixture is obtained; the preparation method comprises the following steps: unfreezing frozen PRP, and mixing the unfrozen PRP with ddH2O to obtain a mixed solution; adding the mixture into the mixed solution, uniformly stirring, and eliminating bubbles to obtain ink; and printing the ink by using a 3D printer, and then placing the ink in a CaCl2 solution for deformation treatment to obtain the PRP combined carboxymethyl chitosan scaffold. Through a 4D printing technology, PPR is changed into a part of printing ink in a stirring manner, and slow release of growth factors in PRP is realized by using a microporous structure of the stent and slow degradation of the stent.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, in particular to a PRP combined with carboxymethyl chitosan scaffold and a preparation method and application thereof. Background Art

[0002] 3D printing technology can prepare three-dimensional scaffolds with high surface area, high porosity and high mechanical strength through digital controllable printing equipment, and construct high-precision material models that perfectly match the defect site. Therefore, it is widely used in medical, smart home, aerospace and other fields. In recent years, with the development of printing technology, time is added as the fourth dimension in three-dimensional space, and programming design related to the time dimension is introduced in the printing design process, that is, 4D printing technology has gradually entered people's field of vision. 4D printing specifically refers to the combination of 3D printing technology and shape memory materials, the initial shape is designed by computer programming, and the 3D printer is used to print and form, which can undergo controllable secondary deformation under the subsequent stimulation of heat, light, electricity, magnetism and even water. At present, 4D printing technology has been widely used in aerospace, smart home, smart textile, automation equipment and other industries. However, because its programming design usually involves complex interactions between various chemical bonds, the preparation process is relatively cumbersome and cannot be further promoted.

[0003] 4D printing technology has been widely used in the field of bone tissue engineering. Ideal biocompatibility, mechanical strength, matching degree with the bone defect area, and the ability of the scaffold to promote bone defect repair have become key indicators for evaluating 4D printed bone tissue engineering biomaterials. Carboxymethyl chitosan, as a derivative of water-soluble chitosan, greatly improves its water solubility by introducing a hydrophilic group -CH2COOH into the chitosan polymer chain. Due to the amino group (-NH 3+), carboxyl (-CH2COO-) and other active groups, and have a structure similar to that of natural extracellular matrix molecules, so it has good biocompatibility and in vivo degradation and absorption. At the same time, carboxymethyl chitosan has a variety of functional activities, and has the characteristics of promoting cell growth and promoting the repair of damaged tissues, and is widely used in the development of absorbable medical devices and medical health products. However, the disadvantage is that the mechanical properties of carboxymethyl chitosan are weak and cannot meet the requirements of bone tissue engineering for biomaterials. In addition, with the increasing number of patients with bone defects caused by trauma, infection, tumor treatment and radiotherapy, the bone defect areas of most patients are not regular and orderly, but present irregular shapes with uneven shapes. Due to the limitations of viscosity and fluidity, the extruded 3D printed scaffolds of ordinary 3D printed carboxymethyl chitosan ink need to be placed on a temperature-controlled flat platform. Therefore, 3D printed carboxymethyl chitosan scaffolds are almost all two-dimensional planar structures, which cannot match the shape of clinical bone defect affected areas. Therefore, it is very important to develop a carboxymethyl chitosan scaffold with high adaptability to the affected area and strong bone repair ability.

[0004] In 1984, Assoian RK et al. first successfully prepared platelet-rich plasma PRP from human blood by centrifugation. PRP is a platelet concentrate rich in growth factors obtained by centrifuging autologous whole blood in a specific centrifugal method, including platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor, fibroblast growth factor, epidermal growth factor and insulin-like growth factor. These growth factors play an important role in the anti-inflammatory response and granulation tissue formation during bone healing. In addition, because PRP is a derivative of autologous blood products, it avoids the body's immune rejection and provides a safer treatment for bone healing. A large number of studies have shown that PRP can promote the repair of bone defects by releasing rich growth factors. At present, local injection of PRP or direct placement of PRP in the affected area is often adopted in clinical practice. This method is often accompanied by the defect of explosive release of growth factors and rapid clearance in a short time. How to efficiently use autologous blood derivatives to better serve the medical industry has become one of the reasons for limiting the development of blood products. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing a PRP combined with carboxymethyl chitosan scaffold to solve the problems raised in the above background technology.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A method for preparing a PRP combined with carboxymethyl chitosan scaffold comprises the following steps:

[0008] After mixing carboxymethyl chitosan powder and hydroxyapatite powder, genipin powder is added and mixed evenly to obtain a mixture;

[0009] The frozen PRP is thawed and mixed with ddH2O to obtain a mixed solution;

[0010] Add the mixture into the mixed liquid, stir evenly, and eliminate bubbles to obtain ink;

[0011] The ink is printed using a 3D printer and then placed in a CaCl2 solution for deformation treatment to obtain the PRP combined with carboxymethyl chitosan scaffold.

[0012] Preferably, the mass ratio of the carboxymethyl chitosan powder to the hydroxyapatite powder is 5:(0.5-1.5).

[0013] Preferably, in the mixed solution, the volume ratio of thawed PRP to ddH2O is (1.2-3.6):(2.4-4.8).

[0014] Preferably, the mass volume ratio of the genipin powder to the mixed solution is (0.1-0.3) g:100 mL.

[0015] Preferably, the mass volume ratio of the carboxymethyl chitosan powder to the mixed solution is (0.8-1.2) g:6 mL.

[0016] Preferably, after printing the ink using a 3D printer, the step of placing the ink in a CaCl2 solution for deformation treatment specifically includes:

[0017] The ink was printed at different initial angles to control the deformation direction of the bracket. The printed bracket was then quickly dried and immersed in a CaCl2 solution to achieve deformation in different directions. The deformed bracket was cross-linked and cured under the slow action of genipin.

[0018] Preferably, the concentration of the CaCl2 solution is 0.05-0.15 mol / L.

[0019] Another object of the embodiment of the present invention is to provide a PRP combined with carboxymethyl chitosan scaffold prepared by the above preparation method.

[0020] Another object of an embodiment of the present invention is to provide an application of the above-mentioned PRP combined with carboxymethyl chitosan scaffold in the preparation of a shape memory material with photothermal conversion properties.

[0021] Another object of the embodiment of the present invention is to provide an application of the above-mentioned PRP combined with carboxymethyl chitosan scaffold in the preparation of bone repair materials.

[0022] The preparation method of the PRP combined with carboxymethyl chitosan scaffold provided in the embodiment of the present invention utilizes the swelling characteristics of carboxymethyl chitosan hydrogel and its ability to shrink under the stimulation of divalent calcium ions, so that the printed material has the characteristics of easy processing and molding, adjustable shape responsiveness, strong structural designability, low price, etc. In addition, the embodiment of the present invention uses 4D printing technology to turn PPR into a part of the printing ink by stirring, and utilizes the microporous structure of the scaffold and the slow degradation of the scaffold to achieve the slow release of the growth factor in PRP; and by adding hydroxyapatite, not only the mechanical properties of the scaffold are improved, but also a three-dimensional scaffold that fits the morphology of the affected area can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A schematic flow chart of a method for preparing a PRP combined with carboxymethyl chitosan scaffold provided in an embodiment of the present invention.

[0024] Figure 2 This is a graph showing the shear-viscosity characterization analysis results of the stents prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0025] Figure 3 It is a graph showing the storage / loss modulus-strain characterization analysis results of the stents prepared in Examples 1-3 of the present invention and Comparative Examples 1-2.

[0026] Figure 4 It is a graph showing the analysis results of the storage / loss modulus-angular frequency characterization of the brackets prepared in Examples 1-3 of the present invention and Comparative Examples 1-2.

[0027] Figure 5 The graph is a viscosity-time characterization analysis result graph of the stents prepared in Examples 1-3 of the present invention and Comparative Examples 1-2.

[0028] Figure 6 This is a diagram showing the fidelity verification results of the stents prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0029] Figure 7 The figure is a swelling characterization result diagram of the stents prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0030] Figure 8 The elastic modulus and compressive strength characterization results of the stents prepared in Examples 1-3 of the present invention and Comparative Examples 1-2 are shown in FIG.

[0031] Fig. 9 The figure is a degradation test result diagram of the stents prepared in Examples 1-3 of the present invention and Comparative Examples 1-2.

[0032] Fig.10 The basic printing deformation display result diagram of the brackets prepared in Examples 1-3 of the present invention and Comparative Example 2 is shown.

[0033] Fig.11 This is a personalized printed deformation display result diagram of the bracket prepared in Example 1 of the present invention.

[0034] Fig.12 The diagram shows the qualitative and quantitative analysis results of the photothermal performance of the brackets prepared in Examples 1-3 of the present invention and Comparative Example 2.

[0035] Fig.13 This is a qualitative and quantitative analysis result diagram of the photothermal performance of the bracket prepared in Example 2 of the present invention under the stimulation of near-infrared light of different powers.

[0036] Fig.14 This is a graph showing the water contact angle characterization analysis results of the stents prepared in Examples 1-3 of the present invention and Comparative Example 2.

[0037] Fig.15 This is a graph showing the experimental results of CCK-8 cytotoxicity detection of the scaffolds prepared in Examples 1-3 of the present invention and Comparative Example 2.

[0038] Fig.16 The staining images of alkaline phosphatase expression in the blank control group, CH group, CHP40 group, and CHP40+NIR group after 7 and 14 days of bone induction.

[0039] Fig.17 Alizarin red staining images of the blank control group, CH group, CHP40 group, and CHP40+NIR group after 28 days.

[0040] Fig.18 Micro-CT reconstructed images of the skull defect area of ​​rats in the blank control group, CH group, CHP40 group, and CHP40+NIR group 4 and 8 weeks after surgery.

[0041] Fig.19 These are the results of He and Masson staining of the skull defect area in the blank control group, CH group, CHP40 group, and CHP40+NIR group of rats 4 and 8 weeks after surgery. DETAILED DESCRIPTION

[0042] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0043] The embodiment of the present invention provides a PRP combined with carboxymethyl chitosan scaffold and a preparation method and application thereof, the purpose of which is to solve the following two technical problems:

[0044] 1. At present, 4D printing technology has gradually emerged in the field of minimally invasive medicine due to its unique shape memory characteristics. 4D printing specifically refers to the combination of 3D printing technology and shape memory materials, the initial shape is designed by computer programming, and the Dd printer is used to print and shape, and controllable secondary deformation can occur under the stimulation of heat, light, electricity, magnetism and even water. At present, general 3D printers on the market can only print regular planar brackets, and the shape memory characteristics of 4D printing materials can make the planar printed brackets undergo secondary deformation according to the preset programming design to obtain various three-dimensional brackets of different shapes. This type of bracket will also meet a wider range of clinical treatment requirements, especially for patients with severe alveolar ridge absorption encountered during oral medical treatment. However, due to the complex interactions between various chemical bonds and the complicated preparation process, the programming design of 4D printing cannot be further promoted. In view of the above problems, the present invention uses the characteristics of carboxymethyl chitosan and PRP that can swell and cross-link in divalent calcium ion solution, as well as the slow cross-linking characteristics of genipin in the mixed ink, to provide a simple and easy 4D printing programming design method. By adjusting the direction of printing, the curling direction of the stent during secondary deformation is controlled to form various curved stents. The slow cross-linking of genipin and carboxymethyl chitosan in the ink is used to achieve the shaping and mechanical strength enhancement after the stent is deformed. At the same time, by adjusting the original shape of the printed stent, the printing of various types of three-dimensional stents can be achieved, such as the "chrysanthemum" stent printed in the embodiment of the present invention. This programming method is suitable for 4D printing of most hydrogel-type stents. One of the purposes of the embodiment of the present invention is to provide a simple and easy-to-understand 4D printing programming design, so that the printing material has the advantages of easy design and easy printing, so that it can better meet the current clinical treatment requirements, and thus it is expected to be used in clinical minimally invasive implant treatment in the future.

[0045] 2. Platelet-rich plasma PRP is a platelet concentrate rich in growth factors obtained by centrifuging whole blood in a specific centrifugal method. PRP contains growth factors such as platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor, fibroblast growth factor, epidermal growth factor and insulin-like growth factor. These growth factors are essential participants in the bone healing process. In addition, because PRP is a derivative of autologous blood products, it avoids the body's immune rejection and provides a safer treatment for bone healing. At present, a large number of studies have shown that PRP can promote the repair of bone defects by releasing rich growth factors. Therefore, PRP has gradually been favored by clinicians in various departments. However, in clinical practice, local injection of PRP or direct placement of PRP in the affected area is often adopted. This method is often accompanied by an explosive release of growth factors, and the suddenly released growth factors will be quickly cleared in a short time. Therefore, it can only play a role in the early stage of bone repair and is not suitable for complex and long-term bone regeneration processes. How to efficiently use PRP to achieve the slow release of blood product derivative growth factors has become one of the key issues to be solved in clinical treatment. The embodiment of the present invention uses 4D printing technology to prepare a carboxymethyl chitosan / hydroxyapatite / PRP (CMCS / HA / PRP) scaffold, turns PPR into a part of the printing ink by stirring, and uses the microporous structure of the scaffold and the slow degradation of the scaffold to achieve the slow release of growth factors in PRP. At the same time, the embodiment of the present invention uses light stimulation to achieve controllable release of PRP by combining with 808nm near-infrared light.

[0046] Specifically, in one embodiment of the present invention, Figure 1 As shown, a method for preparing a PRP combined with carboxymethyl chitosan scaffold is provided, which comprises the following steps:

[0047] S1, mixing carboxymethyl chitosan powder and hydroxyapatite powder, and then adding genipin powder to mix evenly to obtain a mixture;

[0048] S2, thawing the frozen PRP, and mixing it with ddH2O to obtain a mixed solution;

[0049] S3, adding the mixture into the mixed liquid, stirring evenly, and eliminating bubbles to obtain ink;

[0050] S4. After the ink is printed using a 3D printer, it is placed in a CaCl2 solution for deformation treatment to obtain a PRP combined with carboxymethyl chitosan scaffold.

[0051] It should be noted that the extraction method of PRP is as follows:

[0052] Rat-derived PRP: Blood was collected from rat hearts, and the blood was centrifuged at 3000r for 15min for the first time. The supernatant and the part close to the dividing line were taken. The supernatant obtained by the first centrifugation was centrifuged at 3500r for 15min for the second time. The upper 1 / 2 of the depleted PRP was discarded, and the lower 1 / 2 of the PRP was retained. After resuspending by blowing, it was set aside and stored in a -80℃ refrigerator.

[0053] Human PRP: Collect human venous blood, centrifuge it at 240g for 15 minutes, take the supernatant and the part close to the dividing line; centrifuge the supernatant obtained from the first centrifugation at 600g for 15 minutes, discard the upper 1 / 2 of the depleted PRP, retain the lower 1 / 2 of the PRP, resuspend it after blowing for later use, and freeze it in a -80℃ refrigerator.

[0054] In a preferred embodiment of the present invention, the mass ratio of carboxymethyl chitosan powder to hydroxyapatite powder is 5:(0.5-1.5); in the mixed solution, the volume ratio of thawed PRP to ddH2O is (1.2-3.6):(2.4-4.8); the mass volume ratio of genipin powder to the mixed solution is (0.1-0.3) g:100 mL; the mass volume ratio of carboxymethyl chitosan powder to the mixed solution is (0.8-1.2) g:6 mL.

[0055] In a preferred embodiment of the present invention, the step of printing the ink using a 3D printer and then placing the ink in a CaCl2 solution for deformation treatment specifically includes:

[0056] S41. The ink is printed at different initial angles to control the deformation direction of the bracket. The printed bracket is then quickly dried and then immersed in a CaCl2 solution to achieve deformation in different directions. The deformed bracket is cross-linked and cured under the slow action of genipin.

[0057] In practical applications, the structure of the printed bracket can be designed as two parts. The first part (bottom layer) is a thin film structure without printing intervals. The printing path of the second part (upper layer) is set to an interval of 2 mm, and basic printing is performed at initial angles of 0°, 45°, and 90° to control the deformation direction of the bracket.

[0058] It should be noted that 3D printers (Regenovo, China) can be used for 4D stent printing. The specific printing parameters are as follows: printing at 25-30°C, air pressure set to 0.3-0.5 MPa, printing speed set to 8 mm / s, layer height set to 0.4 mm, inner diameter of the print head selected to be 0.41 mm (22G), and stent size set to 20×10 mm.

[0059] In a preferred embodiment of the present invention, the concentration of the CaCl2 solution is 0.05-0.15 mol / L.

[0060] In another embodiment of the present invention, a PRP combined with carboxymethyl chitosan scaffold prepared by the above preparation method is also provided.

[0061] In another embodiment of the present invention, there is also provided an application of the above-mentioned PRP combined with carboxymethyl chitosan scaffold in the preparation of a shape memory material with photothermal conversion properties.

[0062] Another purpose of an embodiment of the present invention is to provide an application of the above-mentioned PRP combined with carboxymethyl chitosan scaffold in the preparation of bone repair materials; specifically, the embodiment of the present invention can achieve controllable release of PRP by light stimulation in combination with 808nm near-infrared light.

[0063] The following embodiments are some specific implementation cases and experimental cases in practical applications of the present invention, but are not limited thereto.

[0064] Example 1: This example provides a method for preparing a PRP combined with carboxymethyl chitosan scaffold, which comprises the following steps:

[0065] S1. After mixing 1 g of carboxymethyl chitosan powder (CMCS) and 0.2 g of hydroxyapatite powder (HA), 0.012 g of genipin powder was added and mixed evenly to obtain a mixture;

[0066] S2, take out the frozen PRP from the -80°C refrigerator and place it in a 37°C water bath to thaw, mix 1.2 mL of the thawed PRP with 4.8 mL of ddH2O to obtain a mixed solution;

[0067] S3, adding the mixture obtained above to the mixed solution, stirring at 200 rpm / min for 15 min by a magnetic stirrer to obtain ink; then, in order to eliminate bubbles, the ink is centrifuged at 3000 rpm for 5 minutes; after the ink is completely uniform and free of bubbles, it is placed in a printing cartridge for standby use; the above ink is recorded as CMCS / HA / PRP ink;

[0068] S4. The above ink was used to print 4D stents using a 3D printer (Regenovo, China); the specific printing parameters were set as follows: printing at 30°C, the air pressure was set to 0.3-0.5MPa, the printing speed was set to 8mm / s, the layer height was set to 0.4mm, the inner diameter of the print head was selected to be 0.41mm (22G), and the stent size was set to 20×10mm. In addition, the printing path was pre-programmed; specifically, the structure of the printed stent was designed into two parts, the first part (bottom layer) was a thin film structure without printing intervals; the printing path of the second part (upper layer) was set to an interval of 2mm, and basic printing was performed at initial angles of 0°, 45°, and 90° to control the deformation direction of the stent. After the printed hydrogel stent was quickly dried at room temperature, the completely dried stent was immersed in a 0.1mol / L CaCl2 solution to achieve deformation in different directions. The deformation direction (angle) of the stent was consistent with the angle of the upper printing path during printing (0°, 45°, 90°). Then, according to this basic deformation rule (printing angle determines deformation direction), different complex structures were designed through the arrangement and combination of three basic printing angles and deformed in 0.1mol / L CaCl2 solution. Finally, the deformed scaffold was cross-linked and cured under the slow action of genipin inside the ink, causing the scaffold to change color, and a PRP combined with carboxymethyl chitosan scaffold with photothermal properties was obtained, which was recorded as CMCS / HA / 20% PRP scaffold, referred to as CHP20 scaffold.

[0069] Example 2: This example provides a method for preparing a PRP combined with carboxymethyl chitosan scaffold. The only difference between this example and Example 1 is that the two use different inks.

[0070] Specifically, the preparation method of the ink used in Example 2 is as follows:

[0071] After mixing 1g of carboxymethyl chitosan powder with 0.2g of hydroxyapatite powder, 0.012g of genipin powder was added and mixed evenly to obtain a mixture; the frozen PRP was taken out from the -80°C refrigerator and placed in a 37°C water bath to thaw, and 2.4mL of thawed PRP was mixed with 3.6mL of ddH2O to obtain a mixed solution; the mixture obtained above was added to the mixed solution, and stirred at 200rpm / min for 15min by a magnetic stirrer to obtain ink; then, in order to eliminate bubbles, the ink was centrifuged at 3000rpm for 5 minutes; after the ink was completely uniform and free of bubbles, it was placed in a printing barrel for use. The PRP combined with carboxymethyl chitosan scaffold prepared in Example 2 is recorded as CMCS / HA / 40%PRP scaffold, referred to as CHP40 scaffold.

[0072] Example 3: This example provides a method for preparing a PRP combined with carboxymethyl chitosan scaffold. The only difference between this example and Example 1 is that the two use different inks.

[0073] Specifically, the preparation method of the ink used in Example 3 is as follows:

[0074] After mixing 1g of carboxymethyl chitosan powder with 0.2g of hydroxyapatite powder, 0.012g of genipin powder was added and mixed evenly to obtain a mixture; the frozen PRP was taken out from the -80°C refrigerator and placed in a 37°C water bath to thaw, and 3.6mL of thawed PRP was mixed with 2.4mL of ddH2O to obtain a mixed solution; the mixture obtained above was added to the mixed solution, and stirred at 200rp m / min for 15min by a magnetic stirrer to obtain ink; then, in order to eliminate bubbles, the ink was placed at a speed of 3000rp m for centrifugation for 5 minutes; after the ink was completely uniform and free of bubbles, it was placed in a printing barrel for standby use. The PRP combined with carboxymethyl chitosan scaffold prepared in Example 3 was recorded as CMCS / HA / 60%PRP scaffold, referred to as CHP60 scaffold.

[0075] Example 4: This example provides a method for preparing a PRP combined with carboxymethyl chitosan scaffold, which comprises the following steps:

[0076] S1, after mixing 0.8 g of carboxymethyl chitosan powder (CMCS) and 0.08 g of hydroxyapatite powder (HA), 0.006 g of genipin powder was added and mixed evenly to obtain a mixture;

[0077] S2, taking out the frozen PRP from the -80°C refrigerator and placing it in a 37°C water bath to thaw, mixing 1.5 mL of the thawed PRP with 4.5 mL of ddH2O to obtain a mixed solution;

[0078] S3, adding the mixture obtained above to the mixed solution, stirring at 200 rpm / min for 15 min by a magnetic stirrer to obtain ink; then, in order to eliminate bubbles, the ink is centrifuged at 3000 rpm for 5 minutes; after the ink is completely uniform and free of bubbles, it is placed in a printing cartridge for standby use; the above ink is recorded as CMCS / HA / PRP ink;

[0079] S4. Use the above ink to print 4D stents using a 3D printer (Regenovo, China); the specific printing parameters are set as follows: printing at 30°C, air pressure set to 0.3-0.5MPa, printing speed set to 8mm / s, layer height set to 0.4mm, the inner diameter of the print head selected is 0.41mm (22G), and the stent size is set to 20×10mm. The printed stent is placed in a 0.05mol / L CaCl2 solution for deformation. Finally, the deformed stent is cross-linked and cured under the slow action of genipin inside the ink, causing the stent to change color, and a PRP combined with carboxymethyl chitosan stent with photothermal properties can be obtained.

[0080] Example 5: This example provides a method for preparing a PRP combined with carboxymethyl chitosan scaffold, which comprises the following steps:

[0081] S1. After mixing 1.2 g of carboxymethyl chitosan powder (CMCS) and 0.36 g of hydroxyapatite powder (HA), 0.018 g of genipin powder was added and mixed evenly to obtain a mixture;

[0082] S2, taking out the frozen PRP from the -80°C refrigerator and placing it in a 37°C water bath to thaw, mixing 3 mL of the thawed PRP with 3 mL of ddH2O to obtain a mixed solution;

[0083] S3, adding the mixture obtained above to the mixed solution, stirring at 200 rpm / min for 15 min by a magnetic stirrer to obtain ink; then, in order to eliminate bubbles, the ink is centrifuged at 3000 rpm for 5 minutes; after the ink is completely uniform and free of bubbles, it is placed in a printing cartridge for standby use; the above ink is recorded as CMCS / HA / PRP ink;

[0084] S4. Use the above ink to print 4D stents using a 3D printer (Regenovo, China); the specific printing parameters are set as follows: printing at 30°C, air pressure set to 0.3-0.5MPa, printing speed set to 8mm / s, layer height set to 0.4mm, the inner diameter of the print head selected is 0.41mm (22G), and the stent size is set to 20×10mm. The printed stent is placed in a 0.15mol / L CaCl2 solution for deformation. Finally, the deformed stent is cross-linked and cured under the slow action of genipin inside the ink, causing the stent to change color, and a PRP combined with carboxymethyl chitosan stent with photothermal properties can be obtained.

[0085] Comparative Example 1: This comparative example provides a method for preparing a printed stent, the only difference between which and Example 1 is that the two use different inks.

[0086] Specifically, the preparation method of the ink used in Comparative Example 1 is as follows:

[0087] 1.2 g of carboxymethyl chitosan powder and 0.012 g of genipin powder were mixed evenly to obtain a mixture; the mixture obtained above was added to 6 mL of ddH2O, and stirred at 200 rpm / min for 15 min by a magnetic stirrer to obtain ink; then, in order to eliminate bubbles, the ink was centrifuged at 3000 rpm for 5 minutes; after the ink was completely uniform and free of bubbles, it was placed in a printing barrel for use. The bracket obtained in Comparative Example 1 is recorded as a CMCS bracket.

[0088] Comparative Example 2: This comparative example provides a method for preparing a printed stent, the only difference between which and Example 1 is that the two methods use different inks.

[0089] Specifically, the preparation method of the ink used in Comparative Example 2 is as follows:

[0090] After mixing 1g of carboxymethyl chitosan powder with 0.2g of hydroxyapatite powder, 0.012g of genipin powder was added and mixed evenly to obtain a mixture; the mixture obtained above was added to 6mL of ddH2O, and stirred at 200rpm / min for 15min by a magnetic stirrer to obtain ink; then, in order to eliminate bubbles, the ink was centrifuged at 3000rpm for 5 minutes; after the ink was completely uniform and free of bubbles, it was placed in a printing barrel for use. The bracket prepared in Comparative Example 2 is recorded as CMCS / HA bracket, referred to as CH bracket.

[0091] Performance and effect test: 1. The CHP20 stent prepared in Example 1, the CHP40 stent prepared in Example 2, the CHP60 stent prepared in Example 3, the CMCS stent prepared in Comparative Example 1, and the CH stent prepared in Comparative Example 2 were subjected to shear-viscosity characterization analysis. The results are as follows: Figure 2 As shown in the figure, it can be seen that all the inks in Examples 1-3 and Comparative Examples 1-2 have good shear thinning properties, ensuring smooth extrusion of the ink under high shear force. Among them, the addition of HA increases the viscosity of the printing ink, but as the PRP content increases, its viscosity continues to decrease.

[0092] 2. The CHP20 stent prepared in Example 1, the CHP40 stent prepared in Example 2, the CHP60 stent prepared in Example 3, the CMCS stent prepared in Comparative Example 1, and the CH stent prepared in Comparative Example 2 were subjected to storage / loss modulus-strain characterization analysis. The results are as follows: Figure 3 As shown; it can be seen from the figure that the inks of Examples 1-3 and Comparative Examples 1-2 remain stable at a strain of 0.5% and can be used for angular frequency scanning analysis.

[0093] 3. The CHP20 stent prepared in Example 1, the CHP40 stent prepared in Example 2, the CHP60 stent prepared in Example 3, the CMCS stent prepared in Comparative Example 1, and the CH stent prepared in Comparative Example 2 were subjected to storage / loss modulus-angular frequency characterization analysis. The results are as follows: Figure 4 As shown; it can be seen from the figure that under a strain of 0.5%, the storage modulus of the ink of Examples 1-3 and Comparative Examples 1-2 is higher than the loss modulus, proving that the printed ink is in a good gel state.

[0094] 4. The CHP20 stent prepared in Example 1, the CHP40 stent prepared in Example 2, the CHP60 stent prepared in Example 3, the CMCS stent prepared in Comparative Example 1, and the CH stent prepared in Comparative Example 2 were subjected to viscosity-time characterization analysis. The results are as follows: Figure 5 As shown in the figure, it can be seen that the addition of HA and PRP increases the thixotropy of the material, which not only ensures the smooth extrusion of the ink, but also increases the stability of the hydrogel scaffold in the static state.

[0095] 5. The fidelity of the CHP20 stent prepared in Example 1, the CHP40 stent prepared in Example 2, the CHP60 stent prepared in Example 3, the CMCS stent prepared in Comparative Example 1, and the CH stent prepared in Comparative Example 2 was verified. The results are as follows: Figure 6 shown; from Figure 6 As can be seen from A and B, a 20×20×0.4mm single-layer array was designed using 3Dmax, and the size of each microbead column in the array was (1.5×0.8×0.4mm). After printing, photos were taken and recorded. Ten points on the array were randomly selected, and the length and width of the microcolumns were calculated using ImageJ software; the results showed that the addition of HA and PRP increased the shape fidelity of the ink, and the scaffold printed by extrusion was closer to the length and width of the set size specifications (1.5×0.8mm).

[0096] VI. The CHP20 stent prepared in Example 1, the CHP40 stent prepared in Example 2, the CHP60 stent prepared in Example 3, the CMCS stent prepared in Comparative Example 1, and the CH stent prepared in Comparative Example 2 were subjected to swelling characterization analysis. The results are as follows: Figure 7 As shown in the figure, it can be seen that the addition of HA reduces the swelling ratio of the CMC S scaffold, which in turn improves the shape fidelity of the scaffold.

[0097] VII. The elastic modulus and compressive strength of the CHP20 stent prepared in Example 1, the CHP40 stent prepared in Example 2, the CHP60 stent prepared in Example 3, the CMCS stent prepared in Comparative Example 1, and the CH stent prepared in Comparative Example 2 were characterized and analyzed. The results are as follows: Figure 8As shown in the figure, compared with the scaffold before cross-linking, Ca 2+ The scaffold cross-linked with genipin has excellent elastic modulus and compressive strength.

[0098] 8. The CHP20 stent prepared in Example 1, the CHP40 stent prepared in Example 2, the CHP60 stent prepared in Example 3, the CMCS stent prepared in Comparative Example 1, and the CH stent prepared in Comparative Example 2 were subjected to degradation tests. The results are as follows: Fig. 9 As shown; it can be seen from the figure that all the scaffolds of Examples 1-3 and Comparative Examples 1-2 slowly degraded within 8 weeks, and the addition of HA slightly reduced the degradation rate of the scaffolds, but the degradation rate of all the scaffolds matched the rate of new bone formation.

[0099] IX. The CHP20 bracket prepared in Example 1, the CHP40 bracket prepared in Example 2, the CHP60 bracket prepared in Example 3, and the CH bracket prepared in Comparative Example 2 were subjected to basic printing deformation display. The results are as follows: Fig.10 As shown; it can be seen from the figure that the deformation direction of all the stents in Examples 1-3 and Comparative Example 2 in 0.1 mol / L calcium chloride solution is consistent with the pre-programmed printing direction of the stent.

[0100] 10. The CHP20 stent prepared in Example 1 was subjected to personalized printing deformation display, and the results were as follows: Fig.11 As shown; it can be seen from the figure that through the combination of basic printing rules, personalized complex structures can be achieved by controlling the strain angle.

[0101] XI. The photothermal performance of the CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, and the CH scaffold prepared in Comparative Example 2 were qualitatively and quantitatively analyzed. The qualitative analysis results are as follows: Fig.12 As shown in the left figure, the quantitative analysis results are Fig.12 As shown in the right figure; it can be seen from the figure that the scaffold after cross-linking with genipin exhibits excellent photothermal conversion efficiency, at 0.75W / cm 2 The photothermal photography and thermal curves under different power levels showed that the addition of HA and PRP had no significant effect on the photothermal conversion efficiency of the scaffold.

[0102] 12. The photothermal performance of the CHP40 scaffold prepared in Example 2 was qualitatively and quantitatively analyzed under near-infrared light (NIR) stimulation of different powers. The qualitative analysis results are as follows: Fig.13 As shown in the left figure, the quantitative analysis results are Fig.13 As shown in the right figure; it can be seen from the figure that 0.25W / cm 2 , 0.5W / cm 2 , 0.75W / cm 2and 1W / cm 2 Under high-power NIR irradiation, the temperatures of the black CHP40 scaffold after cross-linking with genipin can reach 28.1°C, 34.2°C, 39.1°C and 43.3°C respectively.

[0103] Thirteen, the CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, and the CH scaffold prepared in Comparative Example 2 were subjected to water contact angle characterization analysis. The results are as follows: Fig.14 As shown; it can be seen from the figure that the stents prepared in Examples 1-3 and Comparative Example 2 all have excellent hydrophilicity.

[0104] XIV. The CHP20 scaffold prepared in Example 1, the CHP40 scaffold prepared in Example 2, the CHP60 scaffold prepared in Example 3, and the CH scaffold prepared in Comparative Example 2 were subjected to CCK-8 cytotoxicity test and compared with the blank control group (Blank). The results are as follows: Fig.15 As shown in the figure, it can be seen that from the results on days 1, 3, and 7, PRP significantly promoted cell proliferation ability; on the seventh day, the CHP60 scaffold with the highest PRP content showed the strongest cell proliferation ability.

[0105] 15. Under the same experimental conditions, the CH scaffold prepared in Comparative Example 2 was subjected to osteogenic induction experiment, recorded as CH group; the CHP40 scaffold prepared in Example 2 was subjected to osteogenic induction experiment, recorded as CHP40 group; the CHP40 scaffold prepared in Example 2 was subjected to osteogenic induction experiment under near-infrared light (NIR) stimulation, recorded as CHP40+NIR group, and the blank control group (Blank) was used as a comparison; among them, the expression of alkaline phosphatase (ALP) staining in the blank control group (Blank), CH group, CHP40 group, and CHP40+NIR group after 7 and 14 days of osteoinduction is shown in the figure. Fig.16 As shown in the figure, it can be seen that the CHP40 scaffold showed better ALP expression ability than the blank control group and the CH scaffold, and near-infrared can effectively promote the expression of ALP, and the ALP expression of the CHP40+NIR group was the strongest.

[0106] The results of Alizarin Red (AR) staining in the blank control group (Blank), CH group, CHP40 group, and CHP40+NIR group at 28 days are shown in Fig.17 As shown in the figure, it can be seen that the CHP40 scaffold showed better AR expression ability than the blank control group and CH scaffold, and near-infrared can effectively promote AR expression, and the AR expression in the CHP40+NIR group was the strongest.

[0107] Micro-CT reconstructed images of the skull defect area of ​​rats in the blank control group (Blank), CH group, CHP40 group, and CHP40+NIR group 4 and 8 weeks after surgery. Fig.18 As shown in the figure, it can be seen that the recovery of bone defects in rats stimulated by NIR was better than that in rats without stimulation, and the bone regeneration quality of CHP40 group and CHP40+NIR group was better than that of blank control group and CH group.

[0108] The results of He and Masson staining of the skull defect area in the blank control group, CH group, CHP40 group, and CHP40+NIR group 4 and 8 weeks after surgery are shown in Fig.19 As shown; it can be seen from the figure that compared with the blank control group, the new bone formation in the CHP40+NIR group was significantly increased at 4 weeks and 8 weeks; in the Masson staining experiment, the CHP40 group and the CHP40+NIR group showed more collagen formation than the blank control group and the CH group, and near-infrared heating stimulation produced more microvessels.

[0109] Based on the above-mentioned ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above-mentioned description. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A method for preparing a PRP combined with carboxymethyl chitosan scaffold, characterized in that: The following steps are involved: After mixing carboxymethyl chitosan powder and hydroxyapatite powder, genipin powder is added and mixed evenly to obtain a mixture; The frozen PRP is thawed and mixed with ddH2O to obtain a mixed solution; Add the mixture into the mixed liquid, stir evenly, and eliminate bubbles to obtain ink; The ink is printed using a 3D printer and then placed in a CaCl2 solution for deformation treatment to obtain the PRP combined with carboxymethyl chitosan scaffold.

2. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1, characterized in that: The mass ratio of the carboxymethyl chitosan powder to the hydroxyapatite powder is 5:(0.5-1.5).

3. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1, characterized in that: In the mixed solution, the volume ratio of thawed PRP to ddH2O is (1.2-3.6):(2.4-4.8).

4. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1, characterized in that: The mass volume ratio of the genipin powder to the mixed solution is (0.1-0.3) g:100 mL.

5. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1 or 2, characterized in that: The mass volume ratio of the carboxymethyl chitosan powder to the mixed solution is (0.8-1.2) g:6 mL.

6. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1, characterized in that: After the ink is printed using a 3D printer, the step of placing the ink in a CaCl2 solution for deformation treatment specifically includes: The ink was printed at different initial angles to control the deformation direction of the bracket. The printed bracket was then quickly dried and immersed in a CaCl2 solution to achieve deformation in different directions. The deformed bracket was cross-linked and cured under the slow action of genipin.

7. The method for preparing the PRP combined with carboxymethyl chitosan scaffold according to claim 1 or 6, characterized in that: The concentration of the CaCl2 solution is 0.05-0.15 mol / L.

8. A PRP combined with carboxymethyl chitosan scaffold prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the PRP combined with carboxymethyl chitosan scaffold as claimed in claim 8 in the preparation of a shape memory material with photothermal conversion properties.

10. Use of the PRP combined with carboxymethyl chitosan scaffold as claimed in claim 8 in the preparation of bone repair materials.

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